Novel effects of prohibitin 1 expression level on cholesterol and lipid homeostasis.

Prohibitin 1 (PHB1) plays an important role in maintaining liver health and function. The PHB1 level is decreased in patients with various liver diseases. In this study, liver cancer was induced in liver-specific Phb1 knock-out mice, which were then subjected to hepatic gene and metabolomic analysis. The reduced expression of mRNA expression level of Phb1 induced down-regulation of cholesterol and lipid metabolism. This result was confirmed in a cell model. The expression of Hmgcr and Srebp2 in normal cells decreased when they were treated with cholesterol. In HepG2 cells in which the expression of Phb1 was lowered using siPhb1, the mRNA expression of Hmgcr and Srebp2 also decreased when the cells were treated with cholesterol. Furthermore, in the Phb1 knock-out group, the expression of Fasn and Srebp1 related to lipid metabolism increased but the expression of Ldlr decreased. The expression of Cat and Gpx in cells increased when the expression of Phb1 decreased. Altogether, a decreased expression of Phb1 induces down-regulation of cholesterol- and lipid metabolism-related genes and cholesterol homeostasis is not achieved, particularly in a cholesterol-rich environment. The decrease in Phb1 expression causes excessive oxidative stress in cholesterol and lipid metabolism. Therefore, maintaining a normal level of PHB1 expression is crucial for maintaining cholesterol homeostasis in the liver. Thus, PHB1 may become an important target for non-alcoholic fatty liver disease and lipid metabolism in the future.

The transcription factor Mef2d regulates B:T synapse-dependent GC-TFH differentiation and IL-21-mediated humoral immunity.

Communication between CD4 T cells and cognate B cells is key for the former to fully mature into germinal center-T follicular helper (GC-TFH) cells and for the latter to mount a CD4 T cell-dependent humoral immune response. Although this interaction occurs in a B:T synapse-dependent manner, how CD4 T cells transcriptionally regulate B:T synapse formation remains largely unknown. Here, we report that Mef2d, an isoform of the myocyte enhancer factor 2 (Mef2) transcription factor family, is a critical regulator of this process. In CD4 T cells, Mef2d negatively regulates expression of Sh2d1a, which encodes SLAM-associated protein (SAP), a critical regulator of B:T synapses. We found that Mef2d regulates Sh2d1a expression via DNA binding-dependent transcriptional repression, inhibiting SAP-dependent B:T synapse formation and preventing antigen-specific CD4 T cells from differentiating into GC-TFH cells. Mef2d also impeded IL-21 production by CD4 T cells, an important B cell help signaling molecule, via direct repression of the Il21 gene. In contrast, CD4 T cell-specific disruption of Mef2d led to a substantial increase in GC-TFH differentiation in response to protein immunization, concurrent with enhanced SAP expression. MEF2D mRNA expression inversely correlates with human systemic lupus erythematosus (SLE) patient autoimmune parameters, including circulating TFH-like cell frequencies, autoantibodies, and SLEDAI scores. These findings highlight Mef2d as a pivotal rheostat in CD4 T cells for controlling GC formation and antibody production by B cells.

Saturated Fatty Acid-Induced Impairment of Hepatic Lipid Metabolism Is Worsened by Prohibitin 1 Deficiency in Hepatocytes.

Obesity-associated nonalcoholic fatty liver disease (NAFLD) is characterized by excessive intrahepatic lipid accumulation. Despite the increasing prevalence of NAFLD and obesity, the pathogenesis of NAFLD has not yet been clearly elucidated. Prohibitin 1 (PHB1) is mainly expressed in the inner membrane of mitochondria and is known to play an important role in hepatocyte proliferation and lipid metabolism. In this study, we investigated how PHB1 affects lipid metabolism in murine hepatocytes. To reduce the expression of PHB1, Phb1 small interfering RNA was transfected into normal murine hepatocytes (AML12), and the cells were treated with the saturated fatty acid (SFA), palmitic acid (PA), for 24 h. When PHB1 was inhibited, the cell viability decreased by ∼20%, and it was found that it diminished further after PA treatment in both control and peroxisome proliferator-activated receptor gamma (Ppar-γ) knockdown cell groups. Examination of the mRNA expression levels of key enzymes involved in lipid metabolism revealed that PHB1 led to increased stearoyl-coenzyme A desaturase-1 (Scd1) mRNA levels, which leads to an increase in the synthesis of triglycerides (TGs). It also activates the endoplasmic reticulum (ER) stress response through upregulating C/EBP homologous protein (Chop) mRNA levels. PPAR-γ, which has been reported to be upregulated in NAFLD patients, also showed elevated expression. The expression of carnitine palmitoyltransferase 1A, which is involved in the conversion of excess intracellular SFA to fatty acid by catabolism, was downregulated in the PHB1-deficient group. Furthermore, TG synthesis was further promoted by a marked increase in SCD1 mRNA levels, which was further exacerbated by elevated Chop mRNA levels and Ppar-γ disruption. Taken together, PHB1 deficiency led to altered lipid metabolism, resulting in the increased intracellular lipid accumulation and ER stress. These cytotoxic effects were shown to be further exacerbated by excessive PA treatment.

Effects of Amino Acids Supplementation on Lipid and Glucose Metabolism in HepG2 Cells.

Non-alcoholic fatty liver disease and type 2 diabetes are representing symptoms of metabolic syndrome, which is often accompanied with hepatic fat accumulation and insulin resistance. Since liver is the major site of glucose and lipid metabolism, this study aimed to understand the effects of SCAAs and BCAAs supplementations on glucose and lipid metabolism in HepG2 cells. These cells were pretreated with SAMe, betaine, taurine, and BCAA for 24 h, followed by treatments of a high concentration of glucose (50 mM) or palmitic acid (PA, 0.5 mM) for 48 h to simulate high-glucose and high-fat environments. Pretreatment of BCAA and SCAAs inhibited the fat accumulation. At the transcriptional level, glucose and PA treatment led to significant increase of mRNA gluconeogenic enzyme. The mRNA expression level of GLUT2 was decreased by 20% in the SAMe-treated group and inhibited glucose synthesis by reducing the level of gluconeogenic enzyme. After SAMe or BCAA pretreatment, the mRNA expression of lipogenic enzymes was decreased. The PPAR-γ expression was increased after BCAA pretreatment, but SAMe not only downregulated the expression of PPAR-γ, but also inhibited the expression of ChREBP approximately 20% and SREBP-1c decreased by about 15%. Taken together, the effect of SAMe on glucose and lipid metabolism is significant especially on inhibiting hepatic lipogenesis and gluconeogenesis under the metabolic syndrome environment.

The Relationship between Prohibitin 1 Expression, Hepatotoxicity Induced by Acetaminophen, and Hepatoprotection by S-Adenosylmethionine in AML12 Cells.

Prohibitin 1 (Phb1) is a pleiotropic protein, located mainly in the mitochondrial inner membrane and involved in the regulation of cell proliferation and the stabilization of mitochondrial protein. Acetaminophen (APAP) is one of the most commonly used over-the-counter analgesics worldwide. However, at high dose, the accumulation of N-acetyl-p-benzoquinone imine (NAPQI) can lead to APAP-induced hepatotoxicity. In this study, we sought to understand the regulation of mRNA expression in relation to APAP and GSH metabolism by Phb1 in normal mouse AML12 hepatocytes. We used two different Phb1 silencing levels: high-efficiency (HE, >90%) and low-efficiency (LE, 50-60%). In addition, the siRNA-transfected cells were further pretreated with 0.5 mM of S-adenosylmethionine (SAMe) for 24 h before treatment with APAP at different doses (1-2 mM) for 24 h. The expression of APAP metabolism-related and antioxidant genes such as Cyp2e1 and Ugt1a1 were increased during SAMe pretreatment. Moreover, SAMe increased intracellular GSH concentration and it was maintained after APAP treatment. To sum up, Phb1 silencing and APAP treatment impaired the metabolism of APAP in hepatocytes, and SAMe exerted a protective effect against hepatotoxicity by upregulating antioxidant genes.

Lipopolysaccharide-induced innate immune responses are exacerbated by Prohibitin 1 deficiency and mitigated by S-adenosylmethionine in murine macrophages.

Prohibitin 1 (Phb1) is a pleiotropic protein with multiple functions in mammalian cells including cell cycle regulation and mitochondrial protein stabilization. It has been proposed as a potential therapeutic target for a variety of diseases including inflammatory diseases. In this study, we investigated the potential immune-modulatory functions of Phb1 and anti-inflammatory properties of S-adenosylmethionine (SAMe) using macrophages, which play a major role in the innate immune system. The results showed that expressions of Phb1 mRNA and protein were reduced in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (p<0.05). Phb1 knockdown further ameliorated the mRNA expression of pro- and anti-inflammatory cytokines such as TNF-α, IL-1α, IL-1ß, IL-6, and IL10 in LPS-stimulated RAW 264.7 cells. SAMe significantly attenuated LPS-induced inflammatory responses such as IL-1ß, IL-10, Nos2, and NO production in the presence of siPhb1. Luciferase reporter assay was conducted to determine the mechanisms underlying the effects of Phb1 and SAMe on the immune system. The luciferase activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was significantly increased in LPS-treated RAW 264.7 cells. In addition, the luciferase reporter assay showed increased NF-κB activation in Phb1 knockdown RAW 264.7 cells (p<0.1) and SAMe treatment attenuated the NF-κB luciferase activity in Phb1 knockdown RAW 264.7 cells. Based on the results, we concluded that Phb1 possibly modulates the inflammatory response whereas SAMe has an anti-inflammatory effect on Phb1 knockdown macrophage cells. Furthermore, Phb1 expression level has potential properties of affecting on innate immune system by modulating the NF-κB signaling pathway.

Effects of Coffee Extracts with Different Roasting Degrees on Antioxidant and Anti-Inflammatory Systems in Mice.

Coffee roasting affects the taste, color, and aroma of coffee. The Maillard reaction, a major reaction during the roasting process, produces melanoidin, which affects the overall antioxidant capacity and anti-inflammatory effects of coffee. In this experiment, coffee roasting was divided into four degrees: Light, Medium, City, and French. To examine the in vivo antioxidant and anti-inflammatory effects of coffee extracts with different roasting degrees, we used 10-week-old male C57BL/6 mice. Mice were pre-treated with coffee extracts for 10 days by oral gavage (300 mg/Kg.B.W). After the last pre-treatment, lipopolysaccharide (LPS, 15 mg/Kg.B.W) was injected intraperitoneally for immune stimulation. Histopathological analysis showed that hepatic portal vein invasion and liver necrosis were severe in the LPS-treated group. However, these phenomena were greatly ameliorated when mice were pre-treated with Light- or Medium-roasted coffee extracts. Hepatic glutathione level was increased in the French group but decreased in the LPS-stimulated group. When mice were treated with LPS, mRNA expression level of tumor necrosis factor-alpha (TNF-α) was increased, whereas TNF-α expression was significantly reduced in the Light and Medium groups. Treatment with coffee extracts decreased the mRNA expression levels of interleukin 6 (IL-6) in mice stimulated by LPS, regardless of coffee roasting degrees. These effects decreased with the increasing coffee roasting degree. Results of luciferase reporter assay revealed that these effects of coffee extracts were transcriptionally regulated by the NF-κB pathway. Taken together, these results suggest that the roasting degree affects the antioxidant and anti-inflammatory effects of coffee extracts.

Cellular Antioxidant and Anti-Inflammatory Effects of Coffee Extracts with Different Roasting Levels.

During roasting, major changes occur in the composition and physiological effects of coffee beans. In this study, in vitro antioxidant effects and anti-inflammatory effects of Coffea arabica green coffee extracts were investigated at different roasting levels corresponding to Light, Medium, City, and French roast. Total caffeine did not show huge difference according to roasting level, but total chlorogenic acid contents were higher in light roasted coffee extract than other roasted groups. In addition, light roasted coffee extract had the highest antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. To determine the in vitro antioxidant property, coffee extracts were used to treat AML-12 cells. Intracellular glutathione (GSH) concentration and mRNA expression levels of genes related to GSH synthesis were negatively related to roasting levels. The anti-inflammatory effects of coffee extracts were investigated in lipopolysaccharide-treated RAW 264.7 macrophage cells. The cellular antioxidant activity of coffee extracts exhibited similar patterns as the AML-12 cells. The expression of mRNA for tumor necrosis factor-alpha and interleukin-6 was decreased in cells treated with the coffee extracts and the expression decreased with increasing roasting levels. These data suggest that coffee has physiological antioxidant and anti-inflammatory activities and these effects are negatively correlated with roasting levels in the cell models.